Mycoplasmas are a group of microorganisms which are intermediate in size between bacteria and viruses. Included among the human Mycoplasma species which to date have been characterized are Mycoplasma hominis types 1 and 2, Mycoplasma salivarium, Mycoplasma fermentans, Mycoplasma orale types 1 and 2, and Mycoplasma pneumoniae. The species M. hominis type 1 has been recovered from the genitourinary tract, and its frequent occurrence in association with venereal disease, non-bacterial urethritis, cervicitis, and other inflammatory diseases of the genital tract has been reported as well as its association with exudative pharyngitis. M. pulmonis, while not indigenous to man, has been isolated from tissue cultures inoculated with specimens from leukemia patients. M. hominis type 2, which is occasionally isolated from human specimens, has been shown to be identical to a rodent mycoplasma, M. arthritidis.
The rapid identification of mycoplasmas becomes extremely important in initiating the proper treatment of illnesses of which mycoplasmas are the causative agent, especially because they are resistant to many of the antibiotics and chemotherapeutic agents used for bacterial infections.
In recent years, it has been reported that mycoplasmas are present, as contaminants, in tissue cultures such as are used in the metabolic studies of cells or in the propagation of viruses. A prime contaminant has been identified as M. hominis, type 1. The occurrence of mycoplasma in tissue cultures furnishes a potential source for an erroneous interpretation of results, since the interpretation invariably presumes that cultures are devoid of microbial contaminants.
Techniques and methods for isolating, identifying, and inhibiting the growth of mycoplasmas, particularly the human strains, therefore have become important in the preparation and use of tissue cultures.
Mycoplasma pneumoniae is a small prokaryotic parasite of the human respiratory tract and the etiologic agent of primary atypical pneumonia. This pathogen has no cell wall, and requires exogenous cholesterol for the synthesis of plasma membrane and glucose as a carbon and energy source. In tracheal organ cultures the adhesion of viable mycoplasms to the respiratory epithelium is essential for the initiation of infection (cf. Collier et al., Infect. Immun. 3: 694-701, 1971; Hu et al., op. cit. 11: 704-710, 1975; Hu et al. op. cit. 14: 217-224, 1976). Once bound, M. Pneumonia does not penetrate the epithelial surface, but causes extensive damage to the tracheal epithelium, leading to ciliostasis, loss of cilia, and finally, cell death (Collier et al., in Pathogenic Microplasmas, Ciba Foundation Symposium, Jan. 25 to 27, 1972, Elsevier/North-Holland, Amsterdam, p. 307-327 and Carson et al., Infect. Immun. 29: 1117-1124, 1980).
M. pneumoniae also binds in vitro to many other eucaryotic cells, including human colon carcinoma cells (WiDr), human lung fibroblasts (MRC5), HeLa cells, hamster tracheal epithelial cells, spermatozoa, and erythrocytes. Some of these studies suggest that sialylglycoproteins may be receptors for M. pneumoniae, as treatment of the cells with neuraminidase decreases binding (Manchee et al., Br. J. Exp. Pathol. 50: 66-75, 1969; Sobeslavsky et al., J. Bacgeriol. 96: 695-705, 1968; and Barile, M. F. in The Mycoplasmas, Vol. II, Tully et al., Eds., pp 425-464, Academic Press, New York, 1979).
Recent studies suggest that the organism recognizes Neu-Ac.alpha.2- 3Gal.beta.1-4GlcNAc sequences on erythrocytes, as both glycolipids and glycoproteins containing this structure inhibit adhesion of bacteria, cf. Loomes et al., Nature (Lond) 307: 560-563, 1984; and Loomes et al., Infect. Immun. 47: 15-20, 1985. Other studies, however, suggest that glycolipids are not receptors for M. pneumoniae, cf. Gabridge et al., Infect. Immun. 25: 455-459, 1979; and Geary et al., Isr. J. Med. Sci. 23: 462-468, 1987.
A number of prior art workers have provided methods for detecting and identifying mycoplasmas. For example, Cekoric et al. in U.S. Pat. No. 3,668,075 disclose a method for identifying groups of mycoplasmas based on the fact that certain heparinoid compounds selectively inhibit the growth of mycoplasmas in growth media.
Makela et al. in U.S. Pat. No. 4,652,518 disclose a preparation for detecting chlamydial infections using a lipopolysaccharide for Re-lipopolysaccharide mutants of gram-negative bacteria. The polysaccharide is complexed to a carrier molecule to enhance immunological response.
Waters et al. in U.S. Pat. No. 4,632,902 disclose a method for detecting biological activity using a nutrient growth medium which isolates antibiotics and other microbial growth inhibitors during culturing of a microorganism. The growth medium contains an isolating substance which isolates antimicrobial materials during culturing of a microorganism. The isolating substances may be ion exchange resins or non-functional adsorbent resins.
Keller et al. in U.S. Pat. No. 4,543,328 disclose a method for separating bacteria, fungi, and viruses from blood during extracorporeal circulation of the blood with a biocompatible adsorbent. These polymers are blood compatible, and may be polyacrylates, polymethacrylates, crosslinked polystyrenes, cellulose acetate, collodium, and nylon.
Japanese patent 55-31959 discloses a latex for diagnosis of Mycoplasma pneumoniae infectious diseases comprising a Mycoplasma pneumoniae lipid antigen bound with a suspension of latex particles. The lipid antigen may be produced by extracting Mycoplasma fungi bodies with an organic solvent. The Mycoplasma pneumoniae strains are cultured and the fungi bodies are collected and extracted with an organic solvent. A sensitized latex is produced by adding a solution containing lipid antigen into a suspension where latex particles are suspended, and treating the resultant mixture for 2-4 hours. The latex sensitized with lipid antigen may be conserved at 4.degree. C. for longer than one hour by adding a protecting agent such as glycine or dextran and freeze-drying.
Schiefer et al. in Soecialia Aug. 15, 1978, p. 1011, disclose that surface carbohydrate structures can be visualized on Mycoplasma membranes using a cytochemical staining procedure with concanavalin A and iron-dextran complexes. However, there is no disclosure that this staining can be used for diagnostic purposes.